title: Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients.
abstract: BACKGROUND
  Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance.
  
  
  METHODS
  Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing.
  
  
  RESULTS
  We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G > A, MLH1 c.677 + 3A > T, MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, and MSH2 c.1662-2A > C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A > T, MLH1 c.1039-8 T > A, MSH2 c.2459-18delT, and MSH6 c.3439-16C > T).
  
  
  CONCLUSIONS
  In conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.
authors.affiliation: Center for Genomic Medicine, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. tvoh@rh.dk.
authors.forename: Sanne M
authors.initials: SM
authors.lastname: Petersen
authors.forename: Mette
authors.initials: M
authors.lastname: Dandanell
authors.forename: Lene J
authors.initials: LJ
authors.lastname: Rasmussen
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Gerdes
authors.forename: Lotte N
authors.initials: LN
authors.lastname: Krogh
authors.forename: Inge
authors.initials: I
authors.lastname: Bernstein
authors.forename: Henrik
authors.initials: H
authors.lastname: Okkels
authors.forename: Friedrik
authors.initials: F
authors.lastname: Wikman
authors.forename: Finn C
authors.initials: FC
authors.lastname: Nielsen
authors.forename: Thomas V O
authors.initials: TV
authors.lastname: Hansen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000235:genetics*; D003718:Denmark; D005817:Genetic Counseling; D006801:Humans; D007438:Introns; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D022821:RNA Splice Sites; D044465:White People / Q000235:genetics*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D022821:RNA Splice Sites; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein